Synthetic peptides will be employed to investigate the substrate specificities and structures of cyclic AMP-dependent and cyclic GMP-dependent protein kinases. Analogs of synthetic peptides that correspond to amino acid sequences around phosphorylation sites in protein substrates will be evaluated as inhibitors of each enzyme. Peptide inhibitors will be characterized by determining the type of inhibition exhibited, the relative inhibition constants, and the dissociation constants for binding to each kinase. Substrate and inhibitor peptides will be used to investigate the steady-state kinetic mechanism of cyclic GMP-dependent protein kinase. The interaction between the regulatory and the catalytic subunits of cyclic AMP-dependent kinase will be studied using catalytic subunit having peptide substrate or inhibitor bound at its active site. Aryl azide, chloromethyl ketone, or other derivatives of tightly-binding synthetic peptides will be used in an attempt to covalently label the active site of each enzyme. Such compounds may also serve as irreversible inhibitors of protein kinases. Another area of study will be an immunological characterization of phosphorylation sites in known protein substrates of the cyclic AMP-dependent kinase. These studies may provide new compounds for use as pharmacological tools in the study of the physiological roles of these protein kinases in hormone action.